Genetically-based interaction systems are commonly used in scientific research and in commercial and therapeutic applications derived from that research. Current genetically-based interaction systems are severely limited by a fixed level of interaction sensitivity which is either completely xe2x80x9conxe2x80x9d or completely xe2x80x9coffxe2x80x9d (Fields and Song, 1989; Bartel et al., 1993; Gyuris et al., 1993; Mendelsohn and Brent, 1994; Phizicky and Fields, 1995; Bai and Elledge, 1997; Brachmann and Boeke, 1997; Finley and Brent, 1997; Young, 1998). This creates problems related to both the detection of numerous biologically irrelevant interactions, as well as a failure to detect relevant biological interactions. The consequences of this problem may be either a complete inability or prolonged time required to elucidate important biologically relevant interactions, cellular pathways, and potentially related modulatory agents and drugs.
Historically, the first description of a genetic system to detect molecular interactions is the two-hybrid system (Fields and Song, 1989; FIG. 1). This set forth the original concept and practice of detecting protein-protein interactions in Saccharomyces cerevisiae. This original system features detection of an in vivo protein-protein interaction within the nucleus of the yeast cells. These cells were engineered to express the visually detectable bacterial gene lacZ in the presence of an interaction. Basically, the host cells were transformed with an expressible gene coding for a first hybrid protein composed of a DNA binding domain and a first polypeptide. The host cells were additionally transformed with a second hybrid protein consisting of a transcriptional activation domain and a second polypeptide of stable interaction with the first protein fragment. Finally, the cells were also transformed with a lacZ reporter gene containing at least one DNA binding sequence for the DNA binding domain of the first hybrid protein and capable of being transcribed at increased and detectable levels when the transcriptional activation domain of the second hybrid protein was in close proximity. Field and Song demonstrated that when the two hybrid proteins were expressed, levels of the LacZ reporter protein dramatically increased in the host cell. This indicated that the DNA binding domain in the first hybrid protein was binding to the DNA binding sequence of the reporter gene and that the first polypeptide of the first hybrid protein was interacting with the second polypeptide of the second hybrid protein in such a manner as to bring the transcriptional activation domain of the second hybrid protein into proximity of the lacZ gene and thus increase its transcription and subsequent expression.
This basic approach has been employed in all later two-hybrid and three-hybrid systems. Extensions of this work describe such detection capability in nuclear, cytoplasmic, or membrane locations of eukaryotes (Aronheim et al., 1997; Gyuris et al., 1997), as well as in prokaryotes (Bustos and Schleif, 1993; Bunker and Kingston, 1995; Hays et al., 2000). The initial art has also been subsequently extended to include multiple prokaryotic (Bustos and Schleif, 1993; Bunker and Kingston, 1995; Hays et al., 2000) and eukaryotic organisms (other fungal strains, arthropod, plant, and mammalian cells) (e.g. Vasavada et al., 1991; Fearon et al., 1992; Luo et al., 1997; Shoda et al., 2000).
Parallel approaches to genetic molecular interaction detection have been described for detecting protein interactions with RNA and DNA, as well as with small ligands, including peptides and drugs (Li and Herskowitz, 1993; Yang et al., 1995; SenGupta et al., 1996; Brachmann and Boeke, 1997; Young, 1998). All of these systems work on the same basic concept of using the living cell as a means of detecting the interaction between two intracellular molecules.
Genetic molecular detection systems following the original Fields two-hybrid system also usually include the additional feature of genetic selection (Fields and Song, 1989). Selection allows the detection of an interaction by choosing the phenotype of survival; cells containing proteins that do not interact strongly enough or at all are unable to grow, and are no longer considered. The current methods of selection are limited to an xe2x80x9call or nothingxe2x80x9d auxotrophic nutrient, antibiotic selection or other means of affecting survival (Fields and Song, 1989; Gyuris et al., 1993; Bai and Elledge, 1997). Selection yields a great advantage for the various detection systems, since cells containing potentially irrelevant pairs of candidate interacting molecules are eliminated without intervention from the scientist or other automated analysis.
However, the introduction of genetic selection introduced a new and severely limiting aspect to the in vivo genetic molecular detection systems. All current methods of selecting for molecular interactions in vivo must make a priori assumptions about the strength of the interactions that they detect. The system must be constructed such that there is a threshold above which an interaction will be detected, and below which it will not. That is, there is an implicit assumption that very weak or transient interactions are probably less likely to be real or important. Systems are designed to exclude these interactions because, if systems are too sensitive, they will detect too much background. However, if the system is not sensitive at all, important interactions will be missed. Those constructing these systems built them and tested them, and then used the systems with the most reasonable compromise of detection sensitivity. In short, they chose the compositions that yielded, on average, a tolerable background while missing a tolerable number of biologically relevant interactions.
Early crude attempts to overcome this xe2x80x9call or nothingxe2x80x9d threshold of reporting output have included: (a) exposure of yeast to toxic nutrient analogues at sub-lethal concentrations, for example. 3-AT as a histidine synthesis inhibitor (Mangus et al., 1998); and (b) the creation of complicated genetic modifications of the reporter, which gives several different fixed (nonadjustable) levels of detection (James et al., 1996; Finley and Brent, 1997; Serebriiskii et al., 1999). Such complicated modifications include the use of (b.1.) variable numbers of reporter binding sites, (exemplified by the use of multiple LexA binding sites (by, e.g. multiple LexA binding sites for a Leucine reporter as described in Finley and Brent, 1997), for a Leucine reporter as described in Finely and Brent, 1997), and (b.2) variable distance between reporter building site and the transcriptional start site (West et al., 1984).
A feature of current detection systems is the capacity to turn the detection of protein interactions on or off completely by providing for the expression or lack of expression of the two-hybrid library fusion under standard nutrient conditions. Gyuris et al. (1993) found that by being able to express one of the two hybrid proteins at high levels or by being able to limit expression of one such protein completely, it was possible to show in vivo that the presence of both of the hybrid proteins were necessary for activation of the reporter gene; in other words, they added a switch enabling on or off control of one of the interacting components. This control is useful and exerts its effects by modulating reporter activity, but it does not provide for the continuous adjustability of the sensitivity of a two-hybrid protein interaction system. Thus, the Gyuris system further demonstrates the limitation of the prior art; it is either on or off, above or below the same detection threshold set by the reporters chosen when the system was constructed.
The level of reporter gene expression that will result from any given molecule-molecule interaction in a two-hybrid system is uniform for those molecules used in combination with that reporter. The Brent lab first demonstrated this in experiments using a traditional two-hybrid protein-protein interaction system. The experiments showed that output of the quantitative lacZ reporter was directly proportional to the independently determined strength (or Kd) of the protein-protein interaction for the protein fragments used in the hybrid proteins. If the two proteins interacted strongly in vitro, they gave robust expression from the two-hybrid reporters and vice versa. Therefore, they also demonstrated that the output of a given reporter is constant for a given part of interacting proteins. This is now generally accepted, since many publications of genetic molecular interactions include the quantitative reporter output from the interaction system as a relative indication of the strength of the interaction itself (Edwards et al. 1997).
The present invention yields surprising and unexpected advantages relative to earlier systems in providing for adjustability of the sensitivity of such detection systems.
The present invention comprises an improved two-hybrid or three-hybrid detection method and a kit utilizing this method. The method of the current invention may be used with any conventional two-hybrid or three-hybrid methods, including inhibition or competition two-hybrid methods, as well as any future variations of those methods. In all embodiments of the present invention, the sensitivity of a detectable reporter gene in a host cell is continuously adjustable by altering the relative or absolute amounts of interacting molecules provided to the host cell. The method may be used to detect interactions between any types of molecules including, but not limited to, proteins, polypeptides, DNA molecules, RNA molecules, pharmaceutical agents, other biological or chemical agents, and other small molecules or macromolecules. The method may be used to detect interactions in both prokaryotic and eukaryotic organisms or cells. The molecular interactions may occur at various locations, including, but not limited to, extracellular regions, the cell membrane, the cytoplasm, the nuclear membrane, the nucleus, and other intracellular regions.
In a preferred embodiment, the first chimeric gene and the second chimeric gene are introduced into the host cell. The host cell is then subjected to conditions under which a first hybrid protein and a second hybrid protein are expressed in at least sufficient quantities for the detectable reporter gene within the host cell to be activated. The first chimeric gene contains a first exogenously activatable promoter and a sequence encoding the first hybrid protein. The first hybrid protein contains a DNA binding domain capable of binding near the reporter gene and a first interacting polypeptide(bait). The second chimeric gene contains a second exogenously activatable promoter and a sequence encoding a second hybrid protein. The second hybrid protein contains a transcriptional activation domain capable of inducing or increasing transcription of the reporter gene and a second interacting polypeptide(prey). This second polypeptide may be derived from a library.
The sensitivity of the reporter gene may be altered by adding a first and/or second exogenous activator and thus, altering the relative or absolute amounts of the first and/or second hybrid proteins. These alterations affect the activity and thus sensitivity of the report gene. The sensitivity of this activation may be decreased by adding a first exogenous activator capable of activating the first exogenous promoter. This results in increased production of the first hybrid protein and raises its level in the host cell relative to the level of the second hybrid protein. Thus, after this increase, more DNA binding sites of the reporter gene are occupied by the first hybrid proteins for which there is not second hybrid protein available for interaction. Therefore, less of the reporter genes are activated or activation is weaker.
The sensitivity of the reporter activation may be increased by adding a second exogenous activator capable of activating the second exogenous promoter. This results in increased production of the second hybrid protein and raises its level in the host cell relative to the level of the first hybrid protein. Thus, after this increase, more of the DNA binding sites of the reporter gene are occupied by a first hybrid protein that is additionally interacting with a second hybrid protein. Therefore, more of the reporter genes are activated or activation is stronger. Subsequent to the hybrid protein expressions, detectable reporter gene expression is measured and compared to the amount of expression in the absence of any interaction between the first test protein and the second test protein.
A kit utilizing the method of this invention may also be prepared. The kit may comprise any host cell described above, any first or second chimeric genes described above, or any combination thereof. The kit may also contain the first and second exogenous activators and also chemicals or assays for detecting the detectable reporter gene product.